The potassium chloride (KCI) cotransporter (KCC) is abnormally regulated in sickle red blood cells (SS RBC), and contributes to dehydration that potentiates Hb S polymerization, as demonstrated by this laboratory during the previous project period. In humans and mice with sickle cell disease (SCO), pharmacological or genetic inhibition of KCC results in improved SS RBC hydration. This study will test the hypothesis that reduction in KCC gene expression by short interfering RNA (siRNA) will result in reduced KCC activity and lower cellular hemoglobin concentration (CMC) in RBC, to improve sickle phenotype. RNA interference will be produced in RBC precursors by transduction of hematopoietic stem cells (HSC) with lentivirus vectors (LW) coding for short hairpin RNAs (shRNAs) targeting the three KCC isoforms expressed in RBC. In Specific Aim 1, shRNA constructs will be screened for optimal effect and lack of toxicity in HEK 293 cells over-expressing specific isoforms, examining KCC mRNA and protein levels. Selected constructs targeting each KCC isoform will be introduced into a single LW and used to transduce mouse and human HSC to produce a full KCC knockdown. Erythroid cells differentiated in vitro will be analyzed for KCC mRNA and proteins, and RBC volume phenotype (cell volume, CMC). Normal and sickle human HSC will be compared for KCC expression and the effect of KCC knockdown by LW-shRNA. In specific Aim 2, mouse HSC will be transduced by LW containing shRNAs selected to produce optimal KCC knockdown. Transduced HSC will be used to transplant lethally irradiated normal mice for evaluation of resulting hematopoietic and RBC volume phenotype (Hb, WBC, MCV, CHC, RBC density profile, cation content and transport). Knockdowns in mouse sickle HSC transplanted into normal mice will assess the roles of these transporters in SCO. Comparison of lifespan and response to hypoxia of mice transplanted with sickle HSC +/- KCC knockdown will provide measures of safety of shRNA expression in hematopoietic cells and of the potential therapeutic benefit of manipulating transporter expression as a potential adjunct to globin gene therapy for SCO. Lay Summary: The KCI contransporter (KCC) causes dehydration of sickle cells, which worsens their pathology. This project explores whether hydration of sickle cells can be improved by infecting blood stem cells with gene therapy viruses producing RNA molecules that reduce KCC gene expression.